molecules Article Concise Synthesis of Functionalized Cyclobutene Analogues for Bioorthogonal Tetrazine Ligation Jiayu Sun 1,†, Jie Li 1,†, Hongbao Sun 1,†, Chunling Li 2,3,* and Haoxing Wu 1,* 1 Department of Radiology, West China Hospital, Sichuan University, Guo Xue Xiang 37, Chengdu 610041, China; [email protected] (J.S.); [email protected] (J.L.); [email protected] (H.S.) 2 Department of Neurosurgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu 610072, China 3 Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu 610072, China * Correspondence: [email protected] (C.L.); [email protected] (H.W.); Tel.: +86-028-65261615 (H.W.) † These authors contributed equally to this work. Abstract: Novel bioorthogonal tools enable the development of new biomedical applications. Here we report the concise synthesis of a series of aryl-functionalized cyclobutene analogues using com- mercially available starting materials. Our study demonstrates that cyclobutene acts as a small, strained dienophile to generate stable substrates suitable for bioorthogonal tetrazine ligation. Keywords: functionalized cyclobutene analogues; concise synthesis; tetrazine ligation; bioorthogo- nal chemistry 1. Introduction Citation: Sun, J.; Li, J.; Sun, H.; Li, C.; Wu, H. Concise Synthesis of Tetrazine has attracted increasing attention since the emergence of bioorthogonal Functionalized Cyclobutene chemistry [1–4]. Recent clinical trials have highlighted the biomedical usefulness of the Analogues for Bioorthogonal Tetrazine bioorthogonal reaction between tetrazine and dienophiles, an inverse electron demand Ligation. Molecules 2021, 26, 276. Diels–Alder (IEDDA) reaction [5–8]. A wide range of dienophiles, each with different https://doi.org/molecules26020276 advantages and limitations, have been developed as IEDDA substrates for various research purposes. In particular, highly strained trans-cyclooctenes (TCO) can react with tetrazine Academic Editors: Pierre Audebert, very rapidly, enabling a low concentration reaction in vivo in a short time [9–11]. The Jean-Cyrille Hierso and reaction of allyl-substituted TCO with tetrazine leads to a cascade elimination that has Giuseppe Cirillo been widely used in the design of prodrugs [12–15]. Bicyclooctynes (BCN) react rapidly Received: 19 December 2020 with tetrazine to afford pyridazine as a single product, which can facilitate downstream Accepted: 5 January 2021 fluorogenic probe design [16–18]. Cyclopropene units have also emerged as alternative, Published: 8 January 2021 more compact tags that can react quickly with tetrazine and have proven useful in studies of metabolic engineering [19–22]. The strained dienophiles norbornenes and norborna- Publisher’s Note: MDPI stays neu- dienes, although they react less quickly with tetrazine, serve as a handle for covalent tral with regard to jurisdictional clai- biomolecule labeling or offer a click-to-release feature that can be exploited for template- ms in published maps and institutio- promoted turnover amplification, allowing the detection of endogenous oncogenic mi- nal affiliations. croRNAs [23–27]. In addition, the vinyl ether group is small and easy to prepare and can be decorated onto polymers and fluorophores under mild conditions [28–32]. There- fore, the development of novel dienophiles with different features is expected to provide Copyright: © 2021 by the authors. Li- straightforward access to new applications. censee MDPI, Basel, Switzerland. Cyclobutenes, as small and strained dienophiles, may be good mini-tags for labeling This article is an open access article proteins, since their compactness makes it unlikely that they will disturb the proteins’ distributed under the terms and con- physiological functions. In addition, cyclobutene is less volatile than cyclopropene, which ditions of the Creative Commons At- makes the synthesis of mini-tags easier. Despite the advantages of cyclobutene, only the tribution (CC BY) license (https:// synthesis and bioorthogonal properties of alkyl-substituted cyclobutene analogues have creativecommons.org/licenses/by/ been investigated so far [33]. 4.0/). Molecules 2021, 26, 276. https://doi.org/10.3390/molecules26020276 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 13 Molecules 2021, 26, 276 2 of 13 logues have been investigated so far [33]. In this study, we report the concise synthesis of a series of aryl-substituted cyclo- buteneIn thisderivatives study, we from report commercially the concise synthesisavailable of1,3-cyclobutanediol a series of aryl-substituted derivatives. cyclobutene We also derivativessystematically from investigated commercially the available stability 1,3-cyclobutanediol of the derivatives derivatives. and their We alsokinetics system- in aticallybioorthogonal investigated reactions. the stability of the derivatives and their kinetics in bioorthogonal reactions. 2. Results and Discussion 2. ResultsWe used and 1,3-cyclobutanediol Discussion (1) as the starting material for the synthesis of the de- siredWe cyclobutene used 1,3-cyclobutanediol derivatives, and (1) we as theassumed starting that material the two for hydroxyl the synthesis groups of the would desired be cyclobutenedirectly converted derivatives, into a and double we assumed bond or thatfunctional the two groups hydroxyl upon groups reaction would under be directly appro- convertedpriate conditions. into a double Treating bond 1,3-cyclobutanediol or functional groups with upon p reaction-toluenesulfonyl under appropriate chloride condi-(TsCl) tions.and triethylamine Treating 1,3-cyclobutanediol (TEA) yielded th withe mono-toluene-4-sulfonatep-toluenesulfonyl chloride ester (TsCl) 2 in and moderate triethylamine yield. (TEA)After flash yielded column the mono-toluene-4-sulfonate purification, the sulfonate ester ester2 in reacted moderate with yield. a series After of flashp-substituted column purification,thiophenols the(3a– sulfonate3d), forming ester the reacted corresponding with a series thioethers of p-substituted 4a–4d thiophenolsin up to 86% (3a yield.–3d), formingSubsequent the correspondingtosylation and thioetherselimination4a –generated4d in up tofour 86% novel yield. cyclobutene Subsequent derivatives tosylation and(6a– elimination6d) bearing generateda phenyl foursulfide novel moiety cyclobutene useful for derivatives further functionalization (6a–6d) bearing a(Scheme phenyl sulfide1A). Phenolic moiety cyclobutene useful for further derivatives functionalization were prepared (Scheme by slightly1A). Phenolic modifying cyclobutene the above derivativesmentioned weresteps prepared and by slightly by modifyingusing the abovethe mentionedcommercially steps and byavailable using the3-(benzyloxy)cyclobutyl-4-methylbenzenesulfonate commercially available 3-(benzyloxy)cyclobutyl-4-methylbenzenesulfonate (7). We followed a three-step ( process7). We followed(Scheme a1B) three-step to prepare process two cyclobutene (Scheme1B) toanalogues prepare two(11a cyclobutene and 11b) in analoguesoverall yield ( 11a ofand 22– 11b23.5%.) in overallNone of yield the ofintermediates 22–23.5%. None or final of the prod intermediatesucts were volatile, or final and products all were were stored volatile, for ◦ andmonths all were at −20 stored °C. for months at −20 C. SchemeScheme 1.1. SynthesisSynthesis ofof thethe cyclobutenecyclobutene derivatives.derivatives. ((AA)) Synthesis Synthesis of of phenylthio phenylthio cyclobutene cyclobutene deriva- de- rivatives 6a–6d; (B) Synthesis of phenolic cyclobutene derivatives 11a and 11b. (i) TsCl, Et3N, tives 6a–6d;(B) Synthesis of phenolic cyclobutene derivatives 11a and 11b. (i) TsCl, Et3N, DCM, r.t., DCM, r.t., 9 h; (ii) tBuOK, tBuOH, 80 °C, 7 h, 32–86%; (iii) TsCl, Et3N, DCM, r.t., overnight, 55–79%; 9 h; (ii) tBuOK, tBuOH, 80 ◦C, 7 h, 32–86%; (iii) TsCl, Et N, DCM, r.t., overnight, 55–79%; (iv) TFA, t 3 (iv) TFA, DCM, r.t.,t 1 h; (v) BuOK, DMSO, r.t., 1 h, 46–89%; (vi) Cs2CO3◦, DMF, 80 °C, 8 h, 48–70%; DCM, r.t., 1 h; (v) BuOK, DMSO, r.t., 1 h, 46–89%; (vi) Cs2CO3, DMF, 80 C, 8 h, 48–70%; (vii) Pd/C, (vii) Pd/C, EtOH, H2, r.t., overnight; (viii) Et3N, TsCl, DCM, r.t., 20 h, 70–86% for two steps; (ix) EtOH, H , r.t., overnight; (viii) Et N, TsCl, DCM, r.t., 20 h, 70–86% for two steps; (ix) tBuOK, DMSO, tBuOK, DMSO,2 r.t., 1 h, 45–57%. 3TsCl: 4-tosyl chloride; TFA: trifluoroacetic acid. r.t., 1 h, 45–57%. TsCl: 4-tosyl chloride; TFA: trifluoroacetic acid. The synthesized cyclobutene derivatives were then incubated in phos- The synthesized cyclobutene derivatives were then incubated in phosphate-buffered phate-buffered saline (PBS) with 50% N,N-dimethylformamide (DMF) as co-solvent at 37 saline (PBS) with 50% N,N-dimethylformamide (DMF) as co-solvent at 37 ◦C, and their °C, and their stability was monitored over time using liquid chromatography–mass stability was monitored over time using liquid chromatography–mass spectrometry. All cyclobutenesspectrometry. were All relativelycyclobutenes stable were under relatively these buffer stable conditions, under these irrespective buffer conditions, of the phenyl ir- substituent.respective of After the phenyl incubation substituent. for 12 h, After 90% in ofcubation the original for 12 analogues h, 90% of were the original still present, ana- exceptlogues forwere compound still present,11a except(87%) (Figurefor compound1A). There 11a was(87%) slight (Figure degradation 1A). There for was all slight ana- logues,degradation with